In this method, the intestinal lumen of Caenorhabditis elegans (C. elegans) is labeled with a fluorescent fluid-phase marker, Texas Red-dextran. Since dextran conjugates are membrane impermeable, animals fed with it show a red fluorescent signal in the lumen of the intestine. Texas Red-dextran in the lumen is not efficiently endocytosed by intestinal cells and is not effectively transported to the body cavity paracellularly. It is useful to determine whether round-shaped membrane structures are invaginations from the apical membrane or cytoplasmic vesicles. If the barrier function of the intestinal epithelium is impaired, Texas Red-dextran can leak from the intestinal lumen to the body cavity. Therefore, this method can be used to visualize apical membrane morphology in intestinal cells and to investigate the barrier properties of the intestinal epithelium.

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Labeling of the Intestinal Lumen of Caenorhabditis elegans by Texas Red-dextran Feeding

[Abstract]
In this method, the intestinal lumen of Caenorhabditis elegans (C. elegans) is labeled with a fluorescent fluid-phase marker, Texas Red-dextran. Since dextran conjugates are membrane impermeable, animals fed with it show a red fluorescent signal in the lumen of the intestine. Texas Red-dextran in the lumen is not efficiently endocytosed by intestinal cells and is not effectively transported to the body cavity paracellularly. It is useful to determine whether round-shaped membrane structures are invaginations from the apical membrane or cytoplasmic vesicles. If the barrier function of the intestinal epithelium is impaired, Texas Red-dextran can leak from the intestinal lumen to the body cavity. Therefore, this method can be used to visualize apical membrane morphology in intestinal cells and to investigate the barrier properties of the intestinal epithelium.

Incubate the animals for 90 min at room temperature with occasional gentle agitation every 30 min.

Collect the animals by centrifuging at 400 x g for 2 min and discard the supernatant. Wash with 1 ml of egg buffer at least 3 times. Note: Remove as much Texas Red-dextran in the worm suspension as possible.

Add 1 M levamisole hydrochloride to the worm suspension to a final concentration 10~20 mM.

Transfer the worm suspension to a 1.5% agarose pad on a glass slide and cover it with a coverslip.

Although Texas Red-dextran is not efficiently taken up by the intestinal cells in this feeding method, some other apical endocytic markers such as rhodamine-dextran (Mr 40K) and Texas Red BSA are reported to be available to visualize endocytic vesicles (Grant et al., 2001).

Depletion of tight junction components such as claudins (CLC-1-4 in C. elegans) causes the infiltration of dextran conjugates from the intestinal lumen into the body cavity (Asano et al., 2003).

Melt 1.5% agarose in M9 buffer in a microwave oven and transfer it to a glass tube.

Keep the agarose in a molten state by placing the tube in a heat block at 65 °C.

Place a clean glass slide between two glass slides sealed with pieces of labeling tape over both ends.

Drop the 1.5% agarose onto the center glass slide and place another
clean slide on top, perpendicular to the other three slides.

Gently remove the top perpendicular slide after the agarose solidifies.

Use the agarose before it dries up.

Acknowledgments

This work has been supported by the JSPS KAKENHI Grant Number 26291036, the Sumitomo Foundation, the Naito Foundation, and the Mochida Memorial Foundation for Medical and Pharmaceutical Research (to Ken Sato). The protocol has been adapted from Saegusa et al. (2014).

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